Current models for the higher order organization of eukaryotic chromosomes have suggested that the DNA-chromatin fiber is organized into a series of looped domains. A critical feature of the model are the nucleoprotein structures at the boundaries of each loop which organize the chromatin fiber into discrete domains. We have identified and characterized a novel class of chromatin structures, called scs, which appear to correspond to boundaries of higher chromosomal domains. We have shown that the scs- structures provide in vivo sites of action for topo II under circumstances where the topology of the domain is altered by gene activation/de- activation. We have also shown that the scs-structures can insulate against position effects, and will function to block the activity of nearby enhancer elements. The primary goals of the experiments proposed here are 1) to identify and characterize proteins which bind to scs DNA segments in vitro, 2) demonstrate that these proteins are associated with scs in vivo, and that this association is important for scs function, 3) characterize the scs nucleoprotein complex in detail, and 4) develop genetic strategies for analyzing how the scs protein complex functions in vivo.